The present invention generally relates to the isolation of precursor cells and their use in bone and cartilage regeneration procedures and, more particularly, is directed to a method for isolating bone/cartilage precursor cells from a variety of body tissue types utilizing cell surface antigen CD34, other precursor cell surface antigens on CD34+ cells, and other positive and negative cell selection techniques.
Osteogenesis and chondrogenesis are highly complex biological processes having considerable medical and clinical relevance. For example, more than 1,400,000 bone grafting procedures are performed in the developed world annually. Most of these procedures are administered following joint replacement surgeries, or during trauma surgical reconstructions. The success or failure of bone grafting procedures depends largely on the vitality of the site of grafting, graft processing, and in the case of allografts, on immunological compatibility between donor and host. Compatibility issues can largely be negated as an important consideration in the case of autologous grafting procedures, which involve taking bone tissue from one site of the patient for transplantation at another site. While autologous bone grafts are generally successful they do require additional surgery in order to harvest the graft material, and not uncommonly are accompanied by post-operative pain, hemorrhage and infection.
Cartilage regeneration and replacement procedures are perhaps even more problematic. Unlike osteogenesis, chondrogenesis does not typically occur to repair damaged cartilage tissue. Attempts to repair damaged cartilage in any clinically meaningful fashion have met with only limited success. In many cases, the most effective treatment for cartilage damage is prosthetic joint replacement.
These and other difficulties with presently available bone-grafting and cartilage regeneration procedures have prompted intensive investigations into the cellular and molecular bases of osteogenesis and chondrogenesis. Some promising research to date has been in the identification and isolation of bone and cartilage precursor cells from marrow and other tissues.
Early investigations into the complexity of bone marrow demonstrated that lethally irradiated animals could be rescued by marrow transplants, suggesting that bone marrow contained a restorative factor having the capacity to regenerate the entire hematopoietic system. More recent experiments have shown that marrow also has the capacity to regenerate bone and other mesenchymal tissue types when implanted in vivo in diffusion chambers. (See e.g. A. Friedenstein et al. "Osteogenesis in transplants of bone marrow cells." J. Embryol. Exp. Morph. 16, 381-390,1960; M. Owen. "The osteogenic potential of marrow." UCLA Symp. on Mol. and Cell. Biol. 46, 247-255, 1987) Results of this nature have led to the conclusion that bone marrow contains one or more populations of pluripotent cells, known as stem cells, having the capacity to differentiate into a wide variety of different cell types of the mesenchymal, hematopoietic, and stromal lineages.
The process of biological differentiation, which underlies the diversity of cell types exhibited by bone marrow, is the general process by which specialized, committed cell types arise from less specialized, primitive cell types. Differentiation may conveniently be thought of as a series of steps along a pathway, in which each step is occupied by a particular cell type potentially having unique genetic and phenotypic characteristics. In the typical course of differentiation a pluripotent stem cell proceeds through one or more intermediate stage cellular divisions, ending ultimately in the appearance of one or more specialized cell types, such as T lymphocytes and osteocytes. The uncommitted cell types which precede the fully differentiated forms, and which may or may not be true stem cells, are defined as precursor cells.
Although the precise signals that trigger differentiation down a particular path are not fully understood, it is clear that a variety of chemotactic, cellular, and other environmental signals come into play. Within the mesenchymal lineage, for example, mesenchymal stem cells (MSC) cultured in vitro can be induced to differentiate into bone or cartilage in vivo and in vitro, depending upon the tissue environment or the culture medium into which the cells are placed. (See e.g. S Wakitani et al. "Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage" J. Bone and Joint Surg, 76-A, 579-592 (1994); J Goshima, V M Goldberg, and Al Caplan, "The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks" Clin. Orthop. 262, 298-311 (1991); H Nakahara et al. "In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells" Exper. Cell Res. 195, 492-503 (1991)).
Studies of this type have conclusively shown that MSC are a population of cells having the capacity to differentiate into a variety of different cell types including cartilage, bone, tendon, ligament, and other connective tissue types. Remarkably, all distinct mesenchymal tissue types apparently derive from a common progenitor stem cell, viz. MSC. The MSC itself is intimately linked to a trilogy of distinctly differentiating cell types, which include hematopoietic, mesenchymal, and stromal cell lineages. Hematopoietic stem cells (HSC) have the capacity for self-regeneration and for generating all blood cell lineages while stromal stem cells (SSC) have the capacity for self-renewal and for producing the hematopoietic microenvironment.
It is a tantalizing though controversial prospect whether the complex subpopulations of cell types present in marrow (i.e. hematopoietic, mesenchymal, and stromal) are themselves progeny from a common ancestor. The search for ancestral linkages has been challenging for experimentalists. Identifying relatedness among precursor and stem cell populations requires the identification of common cell surface markers, termed "differentiation antigens," many of which appear in a transitory and developmentally-related fashion during the course of differentiation. One group, for example, has reported an ancestral connection among MSC, HSC, and SSC, though later issued a partial retraction (S. Huang & L. Terstappen. "Formation of hematopoietic microenvironment and hematopoietic stem cells from single human bone marrow stem cells" Nature, 360, 745-749, 1992; L. Terstappen & S. Huang. "Analysis of bone marrow stem cell" Blood Cells, 20, 45-63, 1994; E K Waller et al. "The common stem cell hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors" Blood, 85, 2422-2435, 1995). However, studies by another group have demonstrated that murine osteoblasts possess differentiation antigens of the Ly-6 family. That finding is significant in the present context because the Ly-6 antigens are also expressed by cells of the murine hematopoietic lineage. (M. C. Horowitz et al. "Expression and regulation of Ly-6 differentiation antigens by murine osteoblasts" Endocrinology, 135, 1032-1043, 1994). Thus, there may indeed be a close lineal relationship between mesenchymal and hematopoietic cell types which has its origin in a common progenitor. A final answer on this question must await further study.
One of the most useful differentiation antigens for following the course of differentiation in human hematopoietic systems is the cell surface antigen known as CD34. CD34 is expressed by about 1% to 5% of normal human adult marrow cells in a developmentally, stage-specific manner (CI Civin et al., "Antigenic analysis of hematopoiesis. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells" J.Immunol., 133, 157-165, 1984). CD34+ cells are a mixture of immature blastic cells and a small percentage of mature, lineage-committed cells of the myeloid, erythroid and lymphoid series. Perhaps 1% of CD34+ cells are true HSC with the remaining number being committed to a particular lineage. Results in humans have demonstrated that CD34+ cells isolated from peripheral blood or marrow can reconstitute the entire hematopoietic system for a lifetime. Therefore, CD34 is a marker for HSC and hematopoietic progenitor cells.
While CD34 is widely recognized as a marker for hematopoietic cell types, it has heretofore never been recognized as a reliable marker for precursor cells having osteogenic potential in vivo. On the contrary, the prior art has taught that bone precursor cells are not hematopoietic in origin and that bone precursor cells do not express the hematopoietic cell surface antigen CD34 (M W Long, J L Williams, and K G Mann "Expression of bone-related proteins in the human hematopoietic microenvironment" J. Clin. Invest. 86, 1387-1395, 1990; M W Long et al. "Regulation of human bone marrow-derived osteoprogenitor cells by osteogenic growth factors" J. Clin. Invest. 95, 881-887,1995; S E Haynesworth et al. "Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies" Bone, 13, 69-80, 1992).
To date, the most common sources of precursor cells having osteogenic potential have been periosteum and marrow. Many researchers use cells isolated from periosteum for in vitro assays (See e.g. I Binderman et al. "Formation of bone tissue in culture from isolated bone cells" J-Cell Nol. 61, 427-439. 1974). The pioneer of the concept of culturing bone marrow to isolate precursor cells for studying bone and cartilage formation is A. J. Friedenstein. He developed a culture method for isolating and expanding cells (CFU-f) from bone marrow which can form bone (A. J. Friedenstein et al. "The development of fibroblast colonies in monolayer cultures of guinea pig bone marrow and spleen cells" Cell Tiss. Kinet. 3, 393-402, 1970). Others have used Friedenstein's culture system extensively to study the origin of osteoblasts (See e.g. M. Owen, "The origin of bone cells in the postnatal organism" Arthr. Rheum. 23, 1073-1080, 1980). Friedenstein showed that CFU-f cells from marrow will form bone, cartilage, and fibrous tissue when implanted, though CFU-f cells cultured from other sources such as thymus, spleen, peripheral blood, and peritoneal fluid will not form bone or cartilage without an added inducing agent. Friedenstein recently discussed the possible clinical utility of CFU-f and pointed out some obstacles that must be overcome, such as the need for culturing for several passages and developing a method for transplanting the cells (A. J. Friedenstein "Marrow stromal fibroblasts" Calcif Tiss. Int. 56(S): S17, 1995).
Similarly, the most common sources of cartilage precursor cells to date have been periosteum, perichondrium, and marrow. Cells isolated from marrow have also been used to produce cartilage in vivo (S. Wakdani et al. "Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage" J. Bone and Joint Surg, 76A, 579-592, 1994). Periosteal and perichondral grafts have also been used as sources of cartilage precursor cells for cartilage repair (S W O'Driscoll et al. "Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion" J. Bone and Joint Surg. 70A, 1017-1035, 1986; R Coutts et al. "Rib perichondral autografts in full-thickness articular defects in rabbits" Clin. Orthop. Rel. Res. 275, 263-273,1992).
In a series of patents, Caplan et al. disclose a method for isolating and amplifying mesenchymal stem cells (MSC) from marrow. (U.S. Pat. Nos. 4,609,551; 5,197,985; and 5,226,914) The Caplan method involves two basic steps: 1) harvesting marrow and 2) amplifying the MSC contained in the harvested marrow by a 2 to 3 week period of in vitro culturing. This method takes advantage of the fact that a particular culture medium favors the attachment and propagation of MSC over other cell types. In a variation on this basic method, MSC are first selected from bone marrow using specific antibodies against MSC prior to in vitro culturing. (Caplan and Haynesworth; WO 92/22584) The in vitro amplified, marrow-isolated MSC may then be introduced into a recipient at a transplantation repair site. (A. Caplan. "precursor cells" J. Ortho. Res. 9, 641, 1991; S. E. Haynesworth, M. A. Baber, and A. L. Caplan. "Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies," Bone, 13, 69-80, 1992).
The current methods used to isolate precursor cells have a number of drawbacks to consider. First, the methods require that bone marrow or other tissues be harvested. Harvesting bone marrow requires an additional surgical procedure with the appendant possibility of complications from anesthesia, hemorrhage, infection, and post-operative pain. Harvesting periosteum or perichondrium is even more invasive. Second, the Caplan method requires a substantial period of time (2 to 3 weeks) for in vitro culturing of marrow-harvested MSC before the cells can be used in further applications. This additional cell culturing step renders the method time-consuming, costly, and subject to more chance for human error.
Consequently, a need exists for a quicker and simpler method for identifying and isolating precursor cells having osteogenic and chondrogenic potential which can be used for in vivo bone and cartilage regeneration procedures.